Hi Book Team,
I have an essay here for you on cell replacement. If
you think it's ok to both contribute and review, I can
also help with reviewing if needed. (Since I remember
someone asked, I have published in immortalism-related
bioethics (in press), evolutionary psychology and
market research and have a master's equivalent in
biochemistry and cell bio).
Further, I remember sjo was very keen on being a
reviewer for the science corner project that we
cancelled. So we can probably get him to review
something for the book, too.
Also if anything else is needed, be sure to let me
know. What is the status of the book project, going
ahead as planned?
Cheers,
j
John Schloendorn zauberkugel@yahoo.com
==
Cell replacement in future rejuvenation therapies
By John Schloendorn
Contact: Zauberkugel@yahoo.com
Abstract
Effective rejuvenation therapies seem in the process of moving from science fiction to science foreseeable. The key mechanisms of aging have been identified and strategies to remedy them have been drafted. As cell replacement technologies mature, they are revealing the potential to become the central theme of futuristic rejuvenation therapies. In particular, if aging is understood as the accumulation of irreversible cell damage over the life time, then replacing aged cells with cells derived from “young” stem cells theoretically has the potential to functionally rejuvenate our entire bodies.
This essay surveys a range of biomedical strategies to make the full use of cell therapy with engineered stem cells, in order to promote the ambitious goal of human rejuvenation therapies. A comprehensive cell replacement scheme designed to functionally rejuvenate complete human tissues will represent a major change of attitudes in the design and application of curative and preventive medical interventions. A key criterion for such therapies is biomedical sustainability: It must be possible to apply them repeatedly, without any accumulation of deleterious side-effects, so that every cell of the target type can be replaced indefinitely often. It is not completely clear, how many and which cell types will turn out to be amenable to this type of replacement therapy. In all non-replacable cells, much of the hopes for rejuvenation therapies rest on Aubrey de Grey’s SENS proposals to repair the molecular damage in situ. Future rejuvenation therapies should seek to strike the right balance between replacing cells that are hard to repair, and repairing cells that are hard to replace.
Introduction
Educated speculation about the feasibility of extreme human life-extension [1,2] has provoked an ethical debate regarding the desirability of such treatments. Opponents of rejuvenation therapies often appeal to metaphysical concepts of human dignity [3,4] or personal identity [5] that they claim depend on aging and mortality. Proponents, on the other hand, have challenged these arguments [6,7] and argue that the staggering loss inflicted by the aging process on both individuals and societies should outweigh even considerable other concerns. [8,9] A third branch of writers abandoned this point-counterpoint debate and rather began to focus on the constructive exploration of the sociological consequences of future rejuvenation therapies. [10,11]
This essay, on the other hand, seeks to provide a technical account of the hypothetical features of the therapies being debated, without going into too much specialized detail. The issues being addressed include an account of aging and cancer as the result of accumulating cell damage, the potential of stem cells from different adult and embryonic sources as devices of rejuvenation therapy and the novel clinical paradigm of destroying cells that are still healthy, but aged (i.e. have a high probability of becoming diseased).
Aging
Aging has been interpreted as the gradual accumulation of molecular damage inside and outside cells that occurs as a by-product of normal metabolism. [12,13] Steve Austad put it succinctly: “We rust and we cook”, referring to intracellular oxidative lesions and chemical rearrangements of the extracellular matrix, respectively. [14] Efforts to break the aging process down into individual causes argue quite clearly that the intracellular aspects of aging are more numerous, more severe and harder to reverse than the extracellular ones. [15] Certain types of age-related extracellular matrix damage can already be reversed (not just retarded) in humans. For example, the small molecule pharmaceutical ALT-711 can break certain types of extracellular protein cross-linking products and has thereby successfully reduced age-related arterial stiffness in phase II clinical trials. [16]
Thus, there is a potential for cell replacement therapies to reverse most aspects of aging, including the more problematic ones, in one go. In theory, one could get around the need to learn the precise mechanisms and relative importance of the intracellular aspects of aging by simply replacing our aged cells with young ones.
In figure 1, accumulating cell damage over time is represented by shade. The darker the shade of the body, the more damage its cells will carry on average, and the more likely they will develop a particular cellular disease.
A key aspect of age-related cell damage is the complex role of genomic DNA mutations (and similarly also epimutations) in aging and cancer. Aging reflects the increasing probability of cells to develop degenerative diseases due to random functional failures, quite possibly in part due to such mutations [17]. However, tumor suppressor mechanisms are also thought to make a substantial contribution to aging by arresting or removing mutated dividing cells that are prone to form tumors. [18] One outcome of such mechanisms is cell senescence, a terminal arrest of proliferation. Senescent cells are not only metabolically abnormal and partially dysfunctional, but also secrete factors that can actively damage the surrounding tissue. [19] This can be seen as an example of evolutionary antagonistic pleiotropy that confers the advantage of very low cancer rates in early, reproductive life, but the disadvantage of large numbers of senescent cells in late life. [20]
Cancer itself results from the specific failure of regulatory mechanisms that allows cells to proliferate until they form life-threatening tumors. Cancer progression involves the interaction of a number of mutated dividing cells, which are thought to be most often stem cells [21], with a permissive tissue. [22] The competition of clones with particular genomic DNA mutations for the body’s resources is thought to give rise to a microevolutionary process that confers on cancers their aggressive systemic flexibility and allows them to respond in a seemingly intelligent manner to endogenous and exogenous therapeutic initiatives. [23] Before a cancer can become life-threatening in humans, it must typically accumulate multiple mutations in this process that allow it to overcome cell-cycle controls [24], disable apoptosis and senescence pathways [25], escape the immune system [26], acquire angiogenesis and metastasis [27] and survive clinical chemotherapy [28].
As a result, cancer therapy often has to involve high doses of genotoxic agents that can cause rather unspecific cell killing, usually with deleterious side-effects on healthy tissues, such as the immune system [29], brain [30] or reproductive tract [31]. Thus, there are two distinct ways in which cell therapy can benefit cancer medicine: It may act preventively by replacing cells before they get much of a chance to accumulate multiple cancer-promoting mutations, along with the aged tissues that would permit their further transformation, and it may act therapeutically to rescue a patient from the cytotoxic side effects of cancer therapy on healthy tissues, thus allowing for higher chemotherapy dosage. In either case, the replacement of aged cells with young ones from exogenous sources would essentially increase the turnover rate of the tissue and thereby give individual malignant clones less time to accumulate cancer-promoting mutations.
In no longer dividing, post-mitotic tissues such as heart muscle or the neuronal fraction of the brain, the situation again seems different. In particular, cancer does not appear to be an issue in these tissues for two plausible reasons. First, the rate of genomic DNA mutations can be expected to be lower, since a key factor in mutagenesis is DNA replication in the presence of DNA damage [32], and DNA replication does not take place. Second, and more importantly, even when mutations do occur, an individual cell is unlikely to unite many cancer-promoting ones, because in the absence of cell division, the microevolutionary process that drives the accumulation of such mutations cannot occur (Without replication, there can be no evolution). Thus, in post-mitotic tissues, types of molecular damage other than genomic DNA mutations are thought to be particularly important. These include the accumulation of mitochondrial mutations and indigestible intracellular aggregates.
Mitochondrial dysfunction as a consequence of mitochondrial DNA mutations is a prevalent feature of aging [33] and several age-related degenerative diseases [34]. Increasing the rate of mitochondrial mutations leads to premature aging in mice [35] and reducing it by overexpressing a mitochondrial reactive oxygen species-scavenging enzyme can result in considerable murine lifespan extension. [36]
The accumulation of indigestible aggregates, too, seems directly associated with several major age-related diseases, including the neurodegenerative tauopathies [37], atherosclerosis [38] and macular degeneration [39]. Furthermore, lipofuscin, a heterogenic, oxidized lipid and protein mix accumulates with age in post-mitotic tissues and is thought to contribute to general functional decline. [40]
In some post-mitotic tissues, cell replacement could seem technically very challenging. In particular, it might turn out to be undesirable to perform massive replacement of the neurons in the brain, due to the effects this might have on personality. Learning and behavior experiments in animals could assess this question only to a degree, because animals typically lack a human-like personality. Thus, non-cell replacement based means to rejuvenate post-mitotic tissues have been proposed, including the augmentation of their lysosomal catabolism, in order to degrade intracellular aggregates [41] and the obviation of mitochondrial mutations by gene therapy of either the mitochondrion [42], or indeed the nucleus, from where the mitochondrial gene products might be alternatively expressed. [43,44]
All that said, we are ready to take a closer look at figure 1. During normal aging, cellular damage accumulates, ever increasing the probability that enough cells fail to cause a specific disease and death. (Fig. 1A) In order to prevent that, one can attempt to obtain replacement cells by various approaches.
Fig. 1.Effect of replacement cell source on age-related molecular damage
All types of cell damage are collectively represented by shade. During normal development and aging, average cell damage increases until a specific cellular disease causes death (A). Specific cellular diseases can be mended by cell replacement with the individual’s own adult stem cells, but this cannot be expected to greatly reduce the average age-related damage load of the body. Ultimately, ever-increasing numbers of cellular diseases due to ever-increasing levels of molecular damage might overwhelm any such therapy (B). When cellular diseases are repaired with therapeutically cloned cells, most types of damage can be reversed. It is conceivable that this type of cell therapy will remain biomedically sustainable for very long times ©. Cell replacement with embryonic stem cells derived freshly from the germ line by fertilization theoretically has the potential to reverse all types of cell damage (D).
Adult stem cell therapy
Adult stem cell therapy is the utilization of stem cells present in an adult individual, in order to regenerate cell loss in the same or a different individual (Fig. 1B). It can involve direct, signaling-mediated activation (e.g. satellite cells [45]) or cell harvest, in vitro expansion, modification and re-transplantation (e.g. mesenchymal [46], neural stem cells [47]). The major advantage of adult stem cell therapy in this sense is that when an individual’s own cells are used, immunological complications are not to be expected. Thus, effective and technically comparatively straightforward therapies can be developed and have already been developed on the basis of adult stem cell therapy. The most widely used such therapy is bone-marrow transplantation for the treatment of leukemia, which will be discussed below. Thus, adult stem cell therapies are clearly suited to cure various age-related diseases.
However, its utility for true rejuvenation therapies seems questionable, owing to intrinsic limitations. When an individual’s own stem cells are used to regenerate diseased tissues, they can carry over any genetic and other types of molecular damage that were present in them. Only one major cause of aging could plausibly be remedied: During stem cell expansion in vivo or in vitro, intracellular aggregates could get diluted out. It is possible that defective mitochondria cannot similarly get diluted out, since mutant mitochondria are thought to proliferate more efficiently than healthy ones, due to their decreased readiness to be removed by autophagy. [48] Most notably, any genomic DNA mutations present in aged adult stem cells could get amplified by the expansion process and the increased replication of mutated cells may accelerate the evolution of cancer. While many cell types can be affected by such mutations, they have been most strikingly demonstrated in neural stem cells. Multiple genomic loci on different chromosomes accumulate DNA deletions with age in murine neural stem cells, resulting in the loss of homozygosity by old-age in nearly every cell. [49] The accumulation of such deletions in tumor-suppressor genes is thought to be a primary cause of the increasing incidence of and mortality from brain cancers in aged humans [50]. Thus, follow-up studies that monitor cancer incidence and biomarkers of aging after adult stem cell therapy are urgently needed.
Alternatives to the activation of aged patient adult stem cells include transplants from young donors to old recipients, or the long-term storage of adult stem cells harvested at young age. These possibilities could arguably get logistically and technically challenging when scaled up, but deserve being aware of. In sum, it is seems unlikely that adult stem cell therapy can sustain a rejuvenating effect in the long run. Although being very useful to cure specific conditions ”after the fact”, it seems unlikely that this will strongly counteract the age-related increase of the risk for such diseases to occur and recur. In other words, it seems to fail the criterion of biomedical sustainability. Perhaps, perfected adult stem cell therapies could help to achieve the controversial goal of compressing age-related morbidity to the very end of life, without thereby greatly increasing maximum life span. [7]
Therapeutic cloning
Many of these limitations can plausibly be overcome by therapeutic cloning (somatic cell nuclear transfer, SCNT). (Fig. 1C) SCNT involves the insertion of a nucleus from a somatic cell into an enucleated oocyte, which can then be tricked into mitosis and the genesis of a blastocyst, from which embryonic stem cell (ESC) lines can be derived. [51] Although many difficulties remain, ESCs can already be greatly expanded in culture and differentiated into an increasing variety of progenitor cells for therapy. [52,53] Therapeutic cloning was proof-of-principled in mice by the correction of models of severe combined immunodeficiency [54] and Parkinson’s disease [55], while an attempt to cure diabetes failed [56]. Cytoplasmic causes of aging, such as mitochondrial mutations and intracellular aggregates can plausibly be reversed by therapeutic cloning, because the cytoplasm is derived from the oocyte. Genomic DNA methylation, which can be an important cancer-inducing factor [57], is partially reset. [58] But since the genomic DNA base-sequence from the somatic cell persists (excluding shortened telomeres [59]), there is still the potential for carryover of genomic DNA mutations. As argued above, if a post-mitotic cell nucleus is used for cloning, it would be unlikely to carry many cancer-promoting mutations. However, nonsense mutations could get amplified and have undesirable clinical consequences. Functional screening and selection in culture might be able to limit this problem. In sum, it is too early to guess whether true sustainability may be achieved by therapeutic cloning. But the technique can certainly be expected to yield treatments that affect the aging process in many ways.
Therapeutic cloning also has the unique property to confer apparently complete immune compatibility on the replacement cells if a recipient nucleus is used. This would eliminate the need for techniques of long-term immune tolerization (discussed below) that the other methods face. One frequent objection to therapeutic cloning is that contemporary techniques require large numbers of oocytes to succeed even once. Even if the latest technical improvements were used [60], scaling this method up for wide-spread clinical use might well face severe oocyte donor shortages. This need might be met by the differentiation of oocytes from ESCs in vitro, which has been accomplished with mouse and human cells. [61] However, the unconditional functionality of human oocytes derived in this way has yet to be demonstrated by in vitro fertilization.
Embryonic stem cell therapy
An easier and more reliable way to obtain pristine ESCs may be in vitro fertilization (IVF) (Fig 1D). [62] Fertilization can plausibly reverse every cause of aging, since it is the natural process that rejuvenates the next organismal generation from adult cells. Thermodynamically speaking, via ESCs, the germ-line might be used as a permanent reference to restore the information content of the aging host to youthful or even embryonic levels, which would represent true sustainability. [63]
Fertilization-derived ESCs have been appropriately differentiated and engrafted to correct murine models of cellular diseases in a range of tissues, including brain [64], liver [65], heart [66], bone-marrow [67], pancreas [68] and cochlear sensory epithelium [69]. In some cases, however, the efficiency and safety of these processes remain unclear or less than what would be desirable for clinical treatments.
Destructive therapy
In order to achieve the continued suppression of age-related degenerative changes, and indefinite life-extension, it seems very beneficial to regularly undergo a sustainable type of cell replacement therapy. In addition to a way to generate and transplant replenishment cells, any such therapy requires an efficient, selective and safe way to ablate existing aged cells. The first, wild-type generation of cells is probably the most difficult to ablate, because it has few built-in mechanisms to mediate its clinical ablation. In many tissues, stem cell niches must be emptied before efficient engraftment can occur. [70]
Bone-marrow transplants for the clinical treatment of leukemia are usually more efficient when T-lymphocytes are present in the graft that remove host stem cells by recognizing their major histocompatibility complex (MHC) antigens (graft-versus-host, GVH effect). The same effect can effectively eliminate residual leukemic cells (graft-versus-leukemia, GVL effect). [71] Thus, the GVH effect provides the means to the complete cellular replacement of the host hematopoietic system. In order to suppress the subsequent attack of graft T-cells on other host cells (GVH-disease, GVHD), T-cells can be made to die on demand (e.g. after completion of the GVL effect) by fitting them with an inducible suicide gene. This strategy, which has been shown to avert GVHD in mice [72] and men [73] almost a decade ago, should be pursued much more energetically in the clinic.
This existing clinical therapy illustrates a principle upon which sustainable cell replacement therapies might be built. Patient cells can be efficiently and selectively destroyed by T-cells recognizing their MHC antigens and the process can be shut down on demand before it begins to threaten patient survival. Graft cells can then repopulate the empty niches and initiate the regeneration of the entire tissue. In the hematopoietic system this works well in a single step, because we can live for a few days without one. In functionally and structurally more critical solid tissues, the process might have to be very gradual, with each ablation being closely followed by donor cell engraftment and tissue regeneration.
T-cells can also be directed against tissue-specific cell-surface markers. This technology is currently being developed as an anti-cancer strategy [74]. For the purpose of cell replacement, donor cells would have the respective surface marker deleted or carry a different allele against which the engineered T-cells would not react. Thus, different tissues might be turned over at different rates, depending on clinical needs and possibilities, while others may be left alone entirely.
Subsequent generations of graft cells can be made to die on demand more easily by fitting them directly with suicide genes. Promising suicide systems include constitutive herpes tyrosine kinase expression that will induce dividing cell death upon gancyclovir administration, or pro-apoptotic signaling proteins expressed under inducible and/or tissue-specific promoters [75]. Inducer could be administered in initially very low, but increasing dosages to achieve gradual cell ablation. Later generations of cells would switch to different and independent types of inducible suicide systems, until all cells of the first generation have been replaced. Then the cycle could be repeated and sustainability would be established.
Structurally difficult organs, such as cartilage or some epithelia might be inaccessible to a cell-by-cell replacement approach such as the one described above. Here, larger-scale replacement techniques might be employed to complement single-cell based therapies. For example, in some cases whole tissues or organs could be surgically removed and replaced with tissue-engineered constructs [76]. In theory, stem cells for tissue engineering can be derived by any of the methods discussed above [77], thus potentially allowing to control the cell damage levels of a whole replacement organ. Although many organs are still completely inaccessible to this technology, progress is encouraging in clinically relevant areas, such as skin [78], cartilage [79], bone [80] and several urologic epithelia [81]. If needed, cells for tissue engineering could be genetically modified prior to the tissue engineering process as discussed above, to ensure their compatibility with other cell replacement endeavors going on in the patient’s body at the same time.
A further immunological problem that would have to be addressed is graft rejection by the host. Host lymphocytes can be expected to recognize suicide protein fragments, MHCs and any other non-host surface features that the graft cells or T-cells for ablation may bear. [82]
In order to avoid such a host-versus-graft immune reaction, the modification of the host, rather than the graft, is a paradigm that is slowly gaining clinical respectability. Full or partial lymphocyte replacement can induce host tolerance towards allogenic MHC molecules and other foreign antigens, greatly improving graft survival. [83]
Thus, the replacement of the host immune system should take place prior to any other cell therapy efforts, in order to tolerize the host towards any foreign antigens that the subsequent parts of the rejuvenation therapy will employ.
Conclusions
A carefully orchestrated cell replacement regimen using many independent cell ablation and replenishment systems has the potential to cater for the demands of all our different tissues simultaneously, and allow for rejuvenation therapies that remain sustainable indefinitely. According to the present understanding of aging that was meta-reviewed above, this would need to be complemented with techniques to repair potential non-replacable cells and to remove extracellular aggregates and cross-linking products, many of which have already been drafted, in order to effect full functional patient rejuvenation. Certainly, the development of such therapies will face formidable obstacles that could not all be covered here. They include potential side-effects of cytotoxic therapy, the problems of directing the differentiation of embryonic stem cells, control of teratoma formation, a possible hormonal signaling component of aging and the potential appearance of causes of aging that cannot be foreseen today at advanced recipient ages.
The task ahead is daunting and success is uncertain. But given the enormous burden for personal and public well-being that frailty and age-related diseases increasingly pose, much more attention should be devoted to the development of therapies that meet the criterion of biomedical sustainability. We must not let the fear of change and apathy towards new biomedical options discourage the work to fulfill one of humanity’s most ancient dreams. A staggering number of human lives may depend on our initiative.
Acknowledgements
Thanks to Dr. Alexandra Stolzing for her valuable advice and to many other members of the Immortality Institute for the fruitful discussions that inspired much of this work.
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